S-nitrosoglutathione reductase (GSNOR) exerts crucial roles in the homeostasis of nitric oxide (NO) and reactive nitrogen species (RNS) in plant cells through indirect control of S-nitrosation, an important protein post-translational modification in signaling pathways of NO. Using cultivated and wild tomato species, we studied GSNOR function in interactions of key enzymes of reactive oxygen species (ROS) metabolism with RNS mediated by protein S-nitrosation during tomato root growth and responses to salinity and cadmium. Application of a GSNOR inhibitor N6022 increased both NO and S-nitrosothiol levels and stimulated root growth in both genotypes. Moreover, N6022 treatment, as well as S-nitrosoglutathione (GSNO) application, caused intensive S-nitrosation of important enzymes of ROS metabolism, NADPH oxidase (NADPHox) and ascorbate peroxidase (APX). Under abiotic stress, activities of APX and NADPHox were modulated by S-nitrosation. Increased production of H2O2 and subsequent oxidative stress were observed in wild Solanumhabrochaites, together with increased GSNOR activity and reduced S-nitrosothiols. An opposite effect occurred in cultivated S. lycopersicum, where reduced GSNOR activity and intensive S-nitrosation resulted in reduced ROS levels by abiotic stress. These data suggest stress-triggered disruption of ROS homeostasis, mediated by modulation of RNS and S-nitrosation of NADPHox and APX, underlies tomato root growth inhibition by salinity and cadmium stress.
- MeSH
- aldehydoxidoreduktasy metabolismus MeSH
- askorbátperoxidasa metabolismus MeSH
- benzamidy chemie metabolismus farmakologie MeSH
- chlorid sodný farmakologie MeSH
- fyziologický stres MeSH
- kadmium toxicita MeSH
- kořeny rostlin účinky léků růst a vývoj metabolismus MeSH
- NADPH-oxidasy metabolismus MeSH
- nitrosace MeSH
- oxid dusnatý metabolismus MeSH
- oxidační stres účinky léků MeSH
- peroxid vodíku metabolismus MeSH
- pyrroly chemie metabolismus farmakologie MeSH
- reaktivní formy dusíku chemie metabolismus MeSH
- reaktivní formy kyslíku chemie metabolismus MeSH
- regulace genové exprese u rostlin účinky léků MeSH
- rostlinné proteiny metabolismus MeSH
- S-nitrosoglutathion farmakologie MeSH
- S-nitrosothioly metabolismus MeSH
- Solanum lycopersicum účinky léků růst a vývoj metabolismus MeSH
- Solanum růst a vývoj metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
The combination of ultrahigh-resolution mass spectrometry imaging (UHRMSI) and ultrahigh-performance liquid chromatography coupled with tandem mass spectrometry (UHPLC/MS/MS) was used for the identification and the spatial localization of atorvastatin (AT) and its metabolites in rat tissues. Ultrahigh-resolution and high mass accuracy measurements on a matrix-assisted laser desorption/ionization (MALDI)-Orbitrap mass spectrometer allowed better detection of desired analytes in the background of matrix and endogenous compounds. Tandem mass spectra were also used to confirm the identification of detected metabolites in complex matrices. The optimization of sample preparation before imaging experiments included the tissue cryogenic sectioning (thickness 20 μm), the transfer to stainless steel or glass slide, and the selection of suitable matrix and its homogenous deposition on the tissue slice. Thirteen matrices typically used for small molecule analysis, e.g., 2,5-dihydroxybenzoic acid (DHB), 1,5-diaminonaphthalene (DAN), 9-aminoacridine (AA), etc., were investigated for the studied drug and its metabolite detection efficiency in both polarity modes. Particular matrices were scored based on the strength of extracted ion current (EIC), relative ratio of AT molecular adducts, and fragment ions. The matrix deposition on the tissue for the most suitable matrices was done by sublimation to obtain the small crystal size and to avoid local variations in the ionization efficiency. UHPLC/MS profiling of drug metabolites in adjacent tissue slices with the previously optimized extraction was performed in parallel to mass spectrometry imaging (MSI) measurements to obtain more detailed information on metabolites in addition to the spatial information from MSI. The quantitation of atorvastatin in rat liver, serum, and feces was also performed.
- MeSH
- feces chemie MeSH
- játra chemie MeSH
- krysa rodu rattus MeSH
- kyseliny heptylové krev metabolismus MeSH
- potkani Wistar MeSH
- pyrroly krev metabolismus MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice metody MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- tkáňová distribuce fyziologie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Ultrahigh-performance liquid chromatography coupled with high-mass-accuracy tandem mass spectrometry (UHPLC-MS-MS) has been used for elucidation of the structures of oxidation products of atorvastatin (AT), one of the most popular commercially available drugs. The purpose of the study was identification of AT metabolites in rat hepatocytes and comparison with electrochemically generated oxidation products. AT was incubated with rat hepatocytes for 24 h. Electrochemical oxidation of AT was performed by use of a three-electrode off-line system with a glassy carbon working electrode. Three supporting electrolytes (0.1 mol L(-1) H2SO4, 0.1 mol L(-1) HCl, and 0.1 mol L(-1) NaCl) were tested, and dependence on pH was also investigated. AT undergoes oxidation by a single irreversible process at approximately +1.0 V vs. Ag/AgCl electrode. The results obtained revealed a simple and relatively fast way of determining the type of oxidation and its position, on the basis of characteristic neutral losses (NLs) and fragment ions. Unfortunately, different products were obtained by electrochemical oxidation and biotransformation of AT. High-mass-accuracy measurement combined with different UHPLC-MS-MS scans, for example reconstructed ion-current chromatograms, constant neutral loss chromatograms, or exact mass filtering, enable rapid identification of drug-related compounds. β-Oxidation, aromatic hydroxylation of the phenylaminocarbonyl group, sulfation, AT lactone and glycol formation were observed in rat biotransformation samples. In contrast, a variety of oxidation reactions on the conjugated skeleton of isopropyl substituent of AT were identified as products of electrolysis.
- MeSH
- biologický transport MeSH
- biotransformace MeSH
- elektrolýza MeSH
- glykoly chemie metabolismus MeSH
- hepatocyty cytologie účinky léků metabolismus MeSH
- hydroxylace MeSH
- koncentrace vodíkových iontů MeSH
- krysa rodu rattus MeSH
- kultivované buňky MeSH
- kyseliny heptylové chemie metabolismus MeSH
- laktony chemie metabolismus MeSH
- molekulová hmotnost MeSH
- oxidace-redukce MeSH
- pyrroly chemie metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- zvířata MeSH
- Check Tag
- krysa rodu rattus MeSH
- mužské pohlaví MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
BACKGROUND: Using our statin analysis method, it was possible to uncover a significant drop in statin levels (atorvastatin, simvastatin, and metabolites) after extracorporeal LDL-cholesterol elimination (EE) in severe familial hypercholesterolemia (FH). The purpose of this work was to identify the mechanism underlying this drop and its clinical significance as well as to propose measures to optimize a pharmacotherapeutical regimen that can prevent the loss of statins. METHODS: Ultra High Performance Liquid Chromatography (UHPLC) connected to the triple quadrupole MS/MS system was used. Patients. A group of long-term treated patients (3-12 years of treatment) with severe FH (12 patients) and treated regularly by LDL-apheresis (immunoadsorption) or haemorheopheresis (cascade filtration) were included in this study. RESULTS: After EE, the level of statins and their metabolites decreased (atorvastatin before/after LDL-apheresis: 8.83/3.46 nmol/l; before/after haemorheopheresis: 37.02/18.94 nmol/l). A specific loss was found (concentration of atorvastatin for LDL-apheresis/haemorheopheresis: 0.28/3.04 nmol/l in washing fluids; 11.07 nmol/l in filters). To prevent substantial loss of statin concentrations, a pharmacotherapeutic regimen with a longer time interval between the dose of statins and EE is recommended (15 hours). CONCLUSIONS: A specific loss of statins was found in adsorbent columns and filters. The decrease can be prevented by the suggested dosage scheme.
- MeSH
- anticholesteremika krev metabolismus MeSH
- dospělí MeSH
- hemofiltrace metody MeSH
- hyperlipoproteinemie typ II krev farmakoterapie terapie MeSH
- kyseliny heptylové krev metabolismus MeSH
- LDL-cholesterol izolace a purifikace MeSH
- lidé středního věku MeSH
- lidé MeSH
- neparametrická statistika MeSH
- pyrroly krev metabolismus MeSH
- separace krevních složek metody MeSH
- simvastatin krev metabolismus MeSH
- tandemová hmotnostní spektrometrie metody MeSH
- vysokoúčinná kapalinová chromatografie metody MeSH
- Check Tag
- dospělí MeSH
- lidé středního věku MeSH
- lidé MeSH
- mužské pohlaví MeSH
- ženské pohlaví MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
It was shown earlier that 2-pyrrolinodoxorubicin was 500-1000 times more active towards human and mouse cancer cells in vitro than parental doxorubicin. However, the biochemical factors responsible for such a large difference in potency between doxorubicin and 2-pyrrolinodoxorubicin are not clear at the molecular level. To provide this information, we have investigated in cell-free media by biochemical and biophysical methods interactions of both anthracyclines with DNA, effects of these interactions on activity of human topoisomerase II, human Bloom's syndrome helicase and prokaryotic T7 RNA polymerase, and the capability of these drugs to form DNA interstrand cross-links in formaldehyde-free medium. Experiments aimed at understanding the properties of double-helical DNA in the presence of doxorubicin and 2-pyrrolinodoxorubicin revealed only small differences in DNA modifications by these anthracyclines and resulting conformational alterations in DNA. Similarly, the ability of 2-pyrrolinodoxorubicin modifications of DNA to inhibit catalytic activity of topoisomerase II does not differ significantly from that of doxorubicin. On the other hand, we demonstrate that an important factor responsible for the markedly higher antiproliferative potency of DNA modifications by 2-pyrrolinodoxorubicin is capability of these modifications to inhibit downstream cellular processes which process DNA damaged by this drug and involve separation of complementary strands of DNA, such as DNA unwinding by helicases or RNA polymerases. In addition, the results are also consistent with the hypothesis that in particular the capability of 2-pyrrolinodoxorubicin to readily form DNA interstrand cross-links is responsible for inhibition of these processes in the cells treated with this analogue of doxorubicin.
- MeSH
- adukty DNA MeSH
- bezbuněčný systém MeSH
- DNA-topoisomerasy typu II metabolismus MeSH
- DNA metabolismus MeSH
- doxorubicin analogy a deriváty chemie metabolismus farmakologie MeSH
- genetická transkripce MeSH
- hexosaminy chemie MeSH
- inhibitory topoisomerasy II metabolismus farmakologie MeSH
- lidé MeSH
- molekulární struktura MeSH
- myši MeSH
- protinádorové látky farmakologie MeSH
- pyrroly metabolismus farmakologie MeSH
- regulace genové exprese u nádorů účinky léků MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- myši MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Atorvastatin belongs to the group of lipid-lowering drugs known as statins. They significantly reduce the levels of total cholesterol, low-density cholesterol and plasma triglycerides therefore they are widely used in the treatment of hypercholesterolemia. Recently developed methods for the determination of atorvastatin and its metabolites in plasma used SPE (solid phase extraction) or LLE (liquid-liquid extraction) as the sample preparation step. However, both procedures are quite time-consuming and need relatively high volume of solvent/sample, which is impractical for the routine analyses of many biological samples. The aim of this work was to develop and validate more suitable sample preparation method for the determination of atorvastatin and its metabolites in biological samples using MEPS (microextraction by packed sorbent). The optimal conditions of MEPS extraction were using C8 sorbent and only 50 μl of the sample. The analytes were eluted by 100 μl of the mixture of acetonitrile:0.1 M ammonium acetate pH 4.5 (95:5, v:v). The analytical method was validated and demonstrated good linearity (r(2)>0.9990), recovery (89-115%) and intra-day precision (RSD<10%). Total time of the sample preparation was three times shorter (7 min) compared to SPE. The volume of sample was twenty times lower and the volume of solvents about ten times lower compared to SPE. Combination of fast MEPS method together with quick UHPLC-MS/MS was used for the determination of atorvastatin and its two metabolites in serum obtained from patients with familiar hypercholesterolemia.
- MeSH
- anticholesteremika izolace a purifikace metabolismus MeSH
- cholesterol krev MeSH
- extrakce na pevné fázi MeSH
- kyseliny heptylové izolace a purifikace metabolismus MeSH
- lidé MeSH
- pyrroly izolace a purifikace metabolismus MeSH
- reprodukovatelnost výsledků MeSH
- statiny izolace a purifikace metabolismus MeSH
- tandemová hmotnostní spektrometrie MeSH
- triglyceridy krev MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
- validační studie MeSH
